This invention relates to pharmaceutically active compounds which inhibit the vitronectin receptor and are useful for the treatment of osteoporosis.
Mammalian bone is constantly undergoing bone remodeling, which is a dynamic process of bone resorption and bone formation. These processes are mediated by specialized cell types: bone formation is the result of the deposition of mineralized bone by osteoblast cells, and bone resorption is the result of the dissolution of bone matrix by osteoclast cells. Many bone diseases are brought about by an imbalance of bone formation relative to bone resorption. For instance, diseases such as osteoporosis are characterized by a net loss of bone matrix. Thus, agents which inhibit bone resorption are useful for the treatment of such diseases.
An activated osteoclast resorbs bone by attaching to the bone matrix, and secreting proteolytic enzymes, organic acids and protons into the sealed compartment formed between its cell membrane and the bone matrix. The acidic environment and proteolytic enzymes effect the dissolution of bone in the sealed compartment to create pits, or lacuna, in the bone surface, which are apparent when the osteoclast detaches from the bone.
Recent studies have indicated that the attachment of osteoclasts to the bone matrix is mediated through cell surface adhesion receptors called integrins. For instance, Davies, et al., J. Cell Biol., 1989, 109, 1817, disclose that the osteoclast functional antigen, which is implicated in the regulation of bone resorption, is biochemically related to the vitronectin receptor. The vitronectin receptor, or the xcex1vxcex23 integrin, is known to bind to bone matrix proteins, such as osteopontin, bone sialoprotein and thrombospondin, which contain the tri-peptide Arg-Gly-Asp (or RGD) motif. Thus, Horton, et al., Exp. Cell Res. 1991, 195, 368, disclose that RGD-containing peptides and an anti-vitronectin receptor antibody (23C6) inhibit dentine resorption and cell spreading by osteoclasts. In addition, Sato, et al., J. Cell Biol. 1990, 111, 1713 disclose that echistatin, a snake venom peptide which contains the RGD sequence, is a potent inhibitor of bone resorption in tissue culture, and inhibits attachment of osteoclasts to bone. Fisher, et al., Endocrinology 1993, 132, 1411, has further shown that echistatin inhibits bone resorption in vivo in the rat EP 528 587 and 528 586 report substituted phenyl derivatives which inhibit osteoclast mediated bone resorption.
Bondinell, et al., in WO 93/00095 (PCT/US92/05463) and PCT/US93/12436 disclose that certain compounds which have a substituted 6-7 bicyclic ring system are useful for inhibiting the fibrinogen receptor, which is an integrin (xcex1IIbxcex23) protein founds on platelets. Other 6-7 bicyclic ring systems which inhibit the fibrinogen receptor are disclosed by Blackburn et al. in WO 93/08174 (PCT/US92/08788). It has now been discovered that certain appropriately substituted 1,4-benzodiazepine, -benzothiazepine, -benzoxazepine and 2-benzazepine compounds are potent inhibitors of the vitronectin receptor. In particular, it has been discovered that certain such compounds are suprisingly more potent inhibitors of the vitronectin receptor than the fibrinogen receptor and consequently are more useful in the treatment of diseases such as osteoporosis, cancer, atherosclerosis and for inhibiting restenosis of an artery.
This invention comprises compounds of the formula (I) as described hereinafter, which have pharmacological activity for the inhibition of the vitronection receptor and are useful in the treatment of osteoporosis.
This invention is also a pharmaceutical composition comprising a compound according to formula (I) and a pharmaceutically carrier.
This invention is also a method of treating diseases which are mediated by ligands which bind to the vitronectin receptor. In a particular aspect, the compounds of this invention are useful for treating osteoporosis, atherosclerosis, restenosis and cancer.
This invention comprises compounds of formula (I): 
wherein
Xxe2x80x94Xxe2x80x2 is NR1xe2x80x94CH, NC(O)R3xe2x80x94CH, Nxe2x95x90C, CR1xe2x95x90C, CHR1xe2x80x94CH, Oxe2x80x94CH or Sxe2x80x94CH;
R1 is H, C1-6 alkyl or benzyl;
R2 is (CH2)nCO2H;
R3 is H, C1-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl, or C3-6cycloalkyl-C0-6alkyl;
R4 is Wxe2x80x94U, Yxe2x80x94(CHR5)mxe2x80x94U or Zxe2x80x94C(O);
R5 and R6 are independently chosen from H, C1-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl and C3-6cycloalkyl-C0-6alkyl;
m is 1 or 2;
n is 1 or 2;
U is NR1C(O), C(O)NR1, CHxe2x95x90CH, Cxe2x89xa1C, CH2xe2x80x94CH2, Oxe2x80x94CH2, CH2xe2x80x94O or xe2x80x94CH2OCONR1; 
W is
Ra is H, OH, NO2, N(R6)2, CON(R6)2, CH2N(R6)2, or R6HNxe2x80x94C(xe2x95x90NH); 
Y is NH2, NHR6, N(R6)2, C(O)N(R6)2, OH, xe2x95x90Nxe2x80x94OR6, 
Z is
Rd is H, N(R1), C1-4alkyl, CON(R1)2, OH, OR1, or Arxe2x80x94C0-4alkyl;
Re is H, C1-4alkyl, Het-C0-4alkyl or Arxe2x80x94C0-4alkyl;
and pharmaceutically acceptable salts thereof,
provided that R3 is not phenylethyl when R4 is (3-amidino)phenylaminocarbonyl and Xxe2x80x94Xxe2x80x2 is NHxe2x80x94CH.
The compounds of formula (I) inhibit the binding of vitronectin and other RGD-containing peptides to the vitronectin (xcex1vxcex23) receptor. Inhibition of the vitronectin receptor on osteoclasts inhibits osteoclastic bone resorption and is useful in the treatment of diseases wherein bone resorption is associated with pathology, such as osteoporosis.
Suitably, Xxe2x80x94Xxe2x80x2 is NHxe2x80x94CH or CH2xe2x80x94CH.
Preferably, U is NR1CO, CONR1 or CH2OCONR1. More preferably U is NR1CO.
Suitably Ra is hydroxy or amino. Preferably Ra is amino.
Suitably W is 
Preferably, W is 
or (3-amidino)phenyl.
Suitably Y is 
OH or NHR6. Suitably R5 is H, phenyl or C1-6alkyl. Suitably R6 is benzyl, 2-pyridinyl or H.
Suitably Z is 1-piperazinyl or 1-piperidinyl.
Suitably R4 is Yxe2x80x94(CH2)mNCH3CO.
Suitably Re is H or substituted or unsubstituted phenyl, benzyl, 2- or 3-pyridinyl, 2- or 4-pyrimidinyl or 1-, 2- or 3-piperidinyl. When Z is 1-piperazinyl, Re is preferably 2- or 3-pyridinyl or 2- or 4-pyrimidinyl. When Z is piperidinyl, Re is preferably 1-piperidinyl.
Preferably Y is NH2 or pyridinyl.
Preferably n is 1.
Preferably R1 is H, methyl or phenylethyl.
Representative of the novel compounds of this invention are the following:
(xc2x1)-7-[[(6-Amino-2-pyridinyl)amino]carbonyl]-4-methyl-3-oxo-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-8-[[(6-amino-2-pyridinyl)amino]carbonyl]-2-methyl-3-oxo-2,3,4,5-tetrahydro1H-2-benzazepine-4-acetic acid;
(xc2x1)-7-[[(6-amino-3-pyridinyl)amino]carbonyl]-3-oxo-4-(2-phenylethyl)-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-1-Acetyl-7-[[(6-amino-2-pyridinyl)amino]carbonyl]-4-methyl-3-oxo-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-2-methyl-3-oxo-8-[[2-(pyridinyl)carbonyl]amino]-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid;
(xc2x1)-8-[(benzyloxycarbonyl)amino]-2-methyl-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid;
(xc2x1)-7-[[[3-(aminoiminomethyl)phenyl]amino]carbonyl]-4-methyl-3-oxo-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-7-[[[3-(aminocarbonyl)phenyl]amino]carbonyl]-4-methyl-3-oxo-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-4-methyl-3-oxo-7-[1-(4-phenylpiperazinyl)carbonyl]-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-4-methyl-3-oxo-7-[(1-piperazinyl)carbonyl]-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-4-methyl-3-oxo-7-[1-[4-(1-piperidinyl)piperidinyl]carbonyl]-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-4-methyl-3-oxo-7-[1-[4-(2-pyridinyl)piperazinyl]carbonyl]-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-4-methyl-3-oxo-7-[1-[4-(phenylmethyl)piperazinyl]carbonyl]-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-4-methyl-3-oxo-7-[1-[4-(2-pyrimidinyl)piperazinyl]carbonyl]-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
8-[[2S-amino-3-phenylpropanoyl]amino]-2-methyl-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4RS-acetic acid;
(xc2x1)-8-[[[(2-hydroxy-2-phenyl)ethyl]methylamino]carbonyl]-2-methyl-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid;
(xc2x1)-7-[[[N-(2-hydroxyethyl)-N-methyl]amino]carbonyl]-4-methyl-3-oxo-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-8-[[(2-(2-pyridinylamino)acetyl]amino]-2-methyl-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid;
(xc2x1)-4-methyl-3-oxo-7-[[(2-phenylaminoethyl)amino]carbonyl]-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-8-[(2-aminoacetyl)amino]-2-methyl-3-oxo-2,3,4,5-tetrahydro-1h-2-benzazepine-4-acetic acid;
(+/xe2x88x92)-8-[(3-aminopropanoyl)amino]-2-methyl-3-oxo-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid; and
(xc2x1)-4-methyl-3-oxo-7-[[[(3-pyridinyl)methyl]amino]carbonyl]-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
Other compounds useful in the method of inhibiting the vitronectin receptor are:
(xc2x1)-7-[[[3-(Aminoiminomethyl)phenyl]amino]carbonyl]-3-oxo-4-(2-phenylethyl)-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid.
(xc2x1)-7-[1-[4-(2-Methyl-4-pyridinyl)piperazinyl]carbonyl]-4-methyl-3-oxo-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid;
(xc2x1)-3-Oxo-4-(2-phenylethyl)-7-[1-[4-(4-pyridinyl)piperazinyl]carbonyl]-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid; and
(xc2x1)-4-Methyl-3-oxo-7-[1-[4-(4-pyridinyl)piperazinyl]carbonyl]-2,3,4,5-tetrahydro-1H-1,4-benzodiazepine-2-acetic acid.
Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe the compounds of this invention. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Biochen, 158, 9 (1984).
C1-4alkyl as applied herein means an optionally substituted alkyl group of 1 to 4 carbon atoms, and includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl. C1-6alkyl additionally includes pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof. C0-4alkyl and C0-6alkyl additionally indicate that no alkyl group need be present (e.g., that a covalent bond is present).
A substituent on a C1-6 alkyl group, may be on any carbon atom which results in a stable structure, and is available by conventional synthetic techniques. Suitable substituents are C1-4alkyl, OR1, SR1, C1-4alkyl, C1-4alkylsulfonyl, C1-4alkylsulfoxyl, xe2x80x94CN, N(R1)2, CH2N(R1)2, xe2x80x94NO2, xe2x80x94CF3, xe2x80x94CO2Rxe2x80x23, xe2x80x94CON(R1)2, xe2x80x94COR1, xe2x80x94NR1C(O)R1, OH, F, Cl, Br, I, or CF3S(O)rxe2x80x94, wherein r is 0 to 2.
Ar, or aryl, as applied herein, means phenyl or naphthyl, or phenyl or naphthyl substituted by one to three substituents, such as those defined above for alkyl, especially C1-4alkyl, C1-4alkoxy, C1-4alkthio, trifluoroalkyl, OH, F, Cl, Br, I or a 1,2-methylene dioxy group.
Het, or heterocycle, indicates an optionally substituted five or six membered monocyclic ring, or a nine or ten-membered bicyclic ring containing one to three heteroatoms chosen from the group of nitrogen, oxygen and sulfur, which are stable and available by conventional chemical synthesis. Illustrative heterocycles are benzofuryl, benzimidazole, benzopyran, benzothiophene, furan, imidazole, indoline, morpholine, piperidine, piperazine, pyrrole, pyrrolidine, tetrahydropyridine, pyridine, thiazole, thiophene, quinoline, isoquinoline, and tetra- and perhydro- quinoline and isoquinoline. Any accessible combination of up to three substituents on the Het ring, such as those defined above for alkyl that are available by chemical synthesis and are stable are within the scope of this invention.
C3-7cycloalkyl refers to an optionally substituted carbocyclic system of three to seven carbon atoms, which may contain up to two unsaturated carboncarbon bonds. Typical of C3-7cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl and cycloheptyl. Any combination of up to three substituents, such as those defined above for alkyl, on the cycloalkyl ring that is available by conventional chemical synthesis and is stable, is within the scope of this invention.
Certain radical groups are abbreviated herein. t-Bu refers to the tertiary butyl radical, Boc refers to the t-butyloxycarbonyl radical, Fmoc refers to the fluorenylmethoxycarbonyl radical, Ph refers to the phenyl radical, Cbz refers to the benzyloxycarbonyl radical, BrZ refers to the o-bromobenzyloxycarbonyl radical, CIZ refers to the o-chlorobenzyloxycarbonyl radical, Bn refers to the benzyl radical, 4-MBzl refers to the 4-methyl benzyl radical, Me refers to methyl, Et refers to ethyl, Ac refers to acetyl, Alk refers to C1-4alkyl, Nph refers to 1- or 2-naphthyl and cHex refers to cyclohexyl. MeArg is Nxcex1-methyl arginine. Tet refers to 5-tetrazolyl.
Certain reagents are abbreviated herein. DCC refers to dicyclohexylcarbodiimide, DMAP refers to dimethylaminopyridine, DIEA refers to diisopropylethylamine, EDC refers to N-ethyl-Nxe2x80x2(dimethylaminopropyl)-carbodiimide. HOBt refers to 1-hydroxybenzotriazole, THF refers to tetrahydrofuran, DMF refers to dimethyl formamide, NBS refers to N-bromo-succinimide, Pd/C refers to a palladium on carbon catalyst, DPPA refers to diphenylphosphoryl azide, BOP refers to benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate, HF refers to hydrofluoric acid, TEA refers to triethylamine, TFA refers to trifluoroacetic acid, PCC refers to pyridinium chlorochromate.
Compounds of the formula (I) are prepared by the general methods described in Schemes I-III. 
Methyl (xc2x1)-7-carboxy-4-methyl-3-oxo-2,3,4,5-tetrahydro-1H -1,4-benzodiazepine-2-acetate (I-1), prepared as described by Bondinell, et al. (WO 93/00095), is converted to an activated form of the carboxylic acid using, for example, EDC and HOBT or SOCl2, and the activated form is subsequently reacted with an appropriate amine to afford the corresponding amide I-2. Many additional methods for converting a carboxylic acid to an amide are known, and can be found in standard reference books, such as xe2x80x9cCompendium of Organic Synthetic Methodsxe2x80x9d, Vol. I-VI (published by Wiley-Interscience). The methyl ester of I-2 is hydrolyzed using aqueous base, for example, aqueous LiOH in THF or aqueous NaOH in methanol, and the intermediate carboxylate salt is acidified with a suitable acid, for instance TFA or HCl, to afford the carboxylic acid I-3. Alternatively, the intermediate carboxylate salt can be isolated, if desired. 
If the amine partner contains a protecting group, the protecting group can be removed either prior or subsequently to the ester hydrolysis step, using methods suitable for selective deprotection of the specific protecting group employed. Such methods are described in Green, xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d (published by Wiley-Interscience). For example, if the amine partner contains a nitrogen group which is protected by a tert-butoxycarbonyl (BOC) group, such as in compound II-1 (prepared by the general methods described above), the BOC group is removed using acidic conditions, such as TFA in CH2Cl2 or HCl in dioxane, to afford the intermediate ammonium salt II-2. Subsequent ester hydrolysis followed by acidification is accomplished as described in Scheme I to afford II-3. 
Piperidine-containing compounds, such as III-3, can be prepared either from a suitably N-protected piperidine derivative, according to the methods described in Schemes I and II, or from a pyridine precursor, such as III-1. For example, the pyridine subunit of III-1 can be reduced to the corresponding piperidine group by hydrogenation over a suitable catalyst, preferably PtO2, in the presence of acid, such as HCl. The resulting piperidinium salt III-2 is then converted to compound III-3 by the methods described in Scheme I.
The core 6-7 fused ring system is prepared from compounds of the general formula (II): 
wherein R10 is NHR1, CO2H and synthetic equivalents thereof, X and Xxe2x80x2 are as defined for formula (I) and R2 and R3 are as defined in formula (I) with any reactive groups protected. Representative methods for preparing the substituted benzodiazepine nucleus are well known in the art, e.g., Hynes, et al., J. Het. Chem., 1988, 25, 1173; Muller, et al., Helv. Chim. Acta., 1982, 65, 2118; Mori, et al., Heterocycles, 1981, 16, 1491. Similarly, methods for preparing benzazepines, 1,4-benzothiazepines, 1,4-benzoxazepines and 1,4-benzodiazepines are known and are disclosed, for instance, in Bondinell, et al., International Patent Application WO 93/00095.
Representative methods for preparing the benzodiazepine nucleus are given by Schemes IV-VI. A representative method for preparing a benzazepine nucleus is given by Scheme VII. A representative method for preparing a benzothiazepine is given by Scheme VIII. An benzoxazepine nucleus may be prepared in the same manner as Scheme VIII, except substituting a benzyl alcohol for a benzyl thiol. 
The simple tri-substituted benzene starting materials are commercially available or prepared by routine methods well known in the art.
Coupling reagents as used herein denote reagents which may be used to form peptide bonds. Typical coupling methods employ carbodiimides, activated anhydrides and esters and acyl halides. Reagents such as EDC, DCC, DPPA, BOP reagent, HOBt, N-hydroxysuccinimide and oxalyl chloride are typical.
Coupling methods to form peptide bonds are generally well known to the art. The methods of peptide synthesis generally set forth by Bodansky et al., The Practice of Peptide Synthesis, Springer-Verlag, Berlin, 1984, Ali et al. in J. Med. Chem., 29, 984 (1986) and J. Med. Chem., 30, 2291 (1987) are generally illustrative of the technique and are incorporated herein by reference.
Typically, the amine or aniline is coupled via its free amino group to an appropriate carboxylic acid substrate using a suitable carbodiimide coupling agent, such as N,Nxe2x80x2 dicyclohexyl carbodiimide (DCC), optionally in the presence of catalysts such as 1-hydroxybenzotriazole (HOBt) and dimethylamino pyridine (DMAP). Other methods, such as the formation of activated esters, anhydrides or acid halides, of the free carboxyl of a suitably protected acid substrate, and subsequent reaction with the free amine of a suitably protected amine, optionally in the presence of a base, are also suitable. For example, a protected Boc-amino acid or Cbz-amidino benzoic acid is treated in an anhydrous solvent, such as methylene chloride or tetrahydrofuran(THF), in the presence of a base, such as N-methyl morpholine, DMAP or a trialkylamine, with isobutyl chloroformate to form the xe2x80x9cactivated anhydridexe2x80x9d, which is subsequently reacted with the free amine of a second protected amino acid or aniline.
Acid addition salts of the compounds are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric, acetic, trifluoroacetic, maleic, succinic or methanesulfonic. Certain of the compounds form inner salts or zwitterions which may be acceptable. Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine. Cations such as Li+, Na+, K+, Ca++, Mg++ and NH4+ are specific examples of cations present in pharmaceutically acceptable salts.
This invention also provides a pharmaceutical composition which comprises a compound according to formula (I) and a pharmaceutically acceptable carrier. Accordingly, the compounds of formula (I) may be used in the manufacture of a medicament. Pharmaceutical compositions of the compounds of formula (I) prepared as hereinbefore described may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation may be a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
Alternately, these compounds may be encapsulated, tableted or prepared in a emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch. lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia. agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
For rectal administration, the compounds of this invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
The compounds described herein are antagonists of the vitronectin receptor, and are useful for treating diseases wherein the underlying pathology is attributable to ligand or cell which interacts with the vitronectin receptor. For instance, these compounds are useful for the treatment of diseases wherein loss of the bone matrix creates pathology. Thus, the instant compounds are useful for the treatment of ostoeporosis, hyperparathyroidism, Paget""s disease, hypercalcemia of malignancy, osteolytic lesions produced by bone metastasis, bone loss due to immobilization or sex hormone deficiency. The compounds of this invention are also believed to have utility as antitumor, antiinflammatory, anti-angiogenic and anti-metastatic agents, and be useful in the treatment of cancer, atherosclerosis and restenosis.
The peptide is administered either orally or parenterally to the patient, in a manner such that the concentration of drug is sufficient to inhibit bone resorption, or other such indication. The pharmaceutical composition containing the peptide is administered at an oral dose of between about 0.1 to about 50 mg/kg in a manner consistent with the condition of the patient. Preferably the oral dose would be about 0.5 to about 20 mg/kg. For acute therapy, parenteral administration is preferred. An intravenous infusion of the peptide in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. Typically, the parenteral dose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg. The compounds are administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg/kg/day. The precise level and method by which the compounds are administered is readily determined by one routinely skilled in the art by comparing the blood level of the agent to the concentration required to have a therapeutic effect.
The compounds may be tested in one of several biological assays to determine the concentration of compound which is required to have a given pharmacological effect.
Inhibition of Vitronectin Binding
Solid-Phase [3H]-SKandF-107260 Binding to xcex1vxcex23: Human placenta or human platelet xcex1vxcex23 (0.1-0.3 mg/mL) in buffer T (containing 2 mM CaCl2 and 1% octylglucoside) was diluted with buffer T containing 1 mM CaCl2, 1 mM MnCl2, 1 mM MgCl2 (buffer A) and 0.05% NaN3, and then immediately added to 96-well ELISA plates (Corning, New York, N.Y.) at 0.1 mL per well. 0.1-0.2 xcexcg of xcex1vxcex23 was added per well. The plates were incubated overnight at 4xc2x0 C. At the time of the experiment, the wells were washed once with buffer A and were incubated with 0.1 mL of 3.5% bovine serum albumin in the same buffer for 1 hr at room temperature. Following incubation the wells were aspirated completely and washed twice with 0.2 mL buffer A.
Compounds were dissolved in 100% DMSO to give a 2 mM stock solution, which was diluted with binding buffer (15 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, 1 mM MgCl2) to a final compound concentration of 100 xcexcM. This solution is then diluted to the required final compound concentration. Various concentrations of unlabeled antagonists (0.001-100 xcexcM) were added to the wells in triplicates, followed by the addition of 5.0 nM of [3H]-SKandF-107260 (65-86 Ci/mmol).
The plates were incubated for 1 hr at room temperature. Following incubation the wells were aspirated completely and washed once with 0.2 mL of ice cold buffer A in a well-to-well fashion. The receptors were solubilized with 0.1 mL of 1% SDS and the bound [3H]-SKandF-107260 was determined by liquid scintillation counting with the addition of 3 mL Ready Safe in a Beckman LS Liquid Scintillation Counter, with 40% efficiency. Nonspecific binding of [3H]-SKandF-107260 was determined in the presence of 2 xcexcM SKandF-107260 and was consistently less than 1% of total radioligand input. The IC50 (concentration of the antagonist to inhibit 50% binding of [3H]-SKandF-107260) was determined by a nonlinear, least squares curve-fitting routine, which was modified from the LUNDON-2 program. The Ki (dissociation constant of the antagonist) was calculated according to the equation: Ki=IC50/(1+L/Kd), where L and Kd were the concentration and the dissociation constant of [3H]-SKandF-107260, respectively.
Compounds of the present invention inhibit vitronectin binding to SKandF 107260 in the concentration range of 0.1 to 25 micromolar. Preferred compounds inhibit vitronectin binding at a concentration of less than 1 micromolar.
Compounds of this invention are also tested for in vitro and in vivo bone resorption in assays standard in the art for evaluating inhibition of bone formation, such as the pit formation assay disclosed in EP 528 587, which may also be performed using human osteoclasts in place of rat osteoclasts, and the ovarectomized rat model, described by Wronski et al., Cells and Materials 1991, Sup. 1, 69-74.
Parathyroidectomized Rat Model
Each experimental group consists of 5-6 male Sprague-Dawley rats. The rats are parathyroidectomized (by the vendor, Taconic Farms) 7 days prior to use. Twenty four hours prior to use, circulating ionized calcium levels are measured in whole blood immediately after it has been withdrawn by tail venipuncture into heparinized tubes. Rats are included if ionized Ca level (measured with a Ciba-Corning model 634 calcium pH analyzer) is xe2x89xa61.2 mM/L. The rats are then put on a diet of calcium-free chow and deionized water. At the start of the experiment the rats weigh approximately 100 g. Baseline Ca levels are measured and the rats are administered control vehicle (saline) or compound (dissolved in saline) as a single intravenous (tail vein) bolus injection followed immediately by a single subcutaneous injection of either human parathyroid hormone 1-34 peptide (hPTH1-34, dose 0.2 mg/kg in saline/0.1% bovine serum albumen, Bachem, Calif.) or the PTH vehicle. The calcemic response to PTH (and any effect of compound on this response) is measured 2 h after compound/PTH administration.
Rat Ulna Drift Model
Each experimental group consists of 8-10 male Sprague-Dawley or Wistar rats of approximately 30-40 g body weight at the start of the experiment. The agent being tested is administered by an appropriate route as single or multiple daily doses for a period of seven days. Prior to administration of the first dose, the rats are given a single dose of a fluorescent marker (tetracycline 25 mg/kg, or calcein 10 mg/kg) that labels the position of bone forming surfaces at that point in time. After dosing of compound has been completed, the rats are killed and both forelimbs are removed at the elbow, the foot is removed at the ankle and the skin removed. The sample is frozen and mounted vertically on a microtome chuck. Cross sections of the midshaft region of the ulna are cut in the cryostat. The rate of bone resorption is measured morphometrically in the medial-dorsal portion of the cortical bone. The measurement is done as follows: the amount of bone resorbed at the periosteal surface is equal to the distance by which the periosteal surface has advanced towards the fluorescent label which had been incorporated at the endosteal bone formation surface on day zero; this distance is calculated by subtracting the width of bone between the label and the periosteal surface on day 7 from the width on day zero; the resorption rate in microns per day is calculated by dividing the result by 7.
Human Osteoclast Resorption Assay (xe2x80x9cPit Assayxe2x80x9d)
Aliquots of osteoclastoma-derived cell suspensions are removed from liquid nitrogen strorage, warmed rapidly at 37xc2x0 C. and washed xc3x971 in RPMI-1640 medium by centrifugation (1000 rpm, 5 mins at 4xc2x0 C.).
Aspirate the medium and replace it with murine anti-HLA-DR antibody, diluted 1:3 in RPMI-1640 medium. Incubate for 30 mins on ice and mix the cell suspension frequently.
The cells are washed xc3x972 with cold RPMI-1640 by centrifugation (1000 rpm, 5 mins at 4xc2x0 C.) and the cells are transferred to a sterile 15 ml centrifuge tube. The number of mononuclear cells are enumerated in an improved Neubauer counting chamber.
Sufficient magnetic beads (5/mononuclear cell), coated with goat anti-mouse IgG, are removed from their stock bottle and placed into 5 ml of fresh medium (this washes away the toxic azide preservative). The medium is removed by immobilizing the beads on a magnet and is replaced with fresh medium.
The beads are mixed with the cells and the suspension is incubated for 30 mins on ice. The suspension is mixed frequently.
The bead-coated cells are immobilized on a magnet and the remaining cells (osteoclast-rich fraction) are decanted into a sterile 50 ml centrifuge tube.
Fresh medium is added to the bead-coated cells to dislodge any trapped osteoclasts. This wash process is repeated xc3x9710. The bead-coated cells are discarded.
The osteoclasts are enumerated in a counting chamber, using a large-bore disposable plastic pasteur to charge the chamber with the sample.
The cells are pelleted by centrifugation and the density of osteoclasts adjusted to 1.5xc3x97104/ml in EMEM medium, supplemented with 10% fetal calf serum and 1.7 g/liter of sodium bicarbonate.
3 ml aliquots of the cell suspension (per treatment) are decanted into 15 ml centrifuge tubes. The cells are pelleted by centrifugation.
To each tube 3 ml of the appropriate treatment are added (diluted to 50 uM in the EMEM medium). Also included are appropriate vehicle controls, a positive control (87MEM1 diluted to 100 ug/ml) and an isotype control (IgG2a diluted to 100 ug/ml). Incubate at 37xc2x0 C. for 30 mins.
0.5 ml aliquots of the cells are seeded onto sterile dentine slices in a 48-well plate and incubated at 37xc2x0 C. for 2 hours. Each treatment is screened in quadruplicate.
The slices are washed in six changes of warm PBS (10 ml/well in a 6-well plate) and then placed into fresh treatment or control. Incubate at 37xc2x0 C. for 48 hours. tartrate resistant acid phosphatase (trap) procedure (selective stain for cells of the osteoclast lineage).
The slices are washed in phosphate buffered saline and fixed in 2% gluteraldehyde (in 0.2M sodium cacodylate) for 5 mins.
They are washed in water and incubated in TRAP buffer for 5 mins at 37xc2x0 C.
Following a wash in cold water they are incubated in cold acetate buffer/fast red garnet for 5 mins at 4xc2x0 C.
Excess buffer is aspirated, and the slices are air dried following a wash in water.
The TRAP positive osteoclasts are enumerated by bright-field microscopy and are then removed from the surface of the dentine by sonication.
Pit volumes are determined using the Nikon/Lasertec ILM21W confocal microscope.
Inhibition of RGD-Mediated GPIIB-IIIA Binding
Purification of GPIIb-IIIa
Ten units of outdated, washed human platelets (obtained from Red Cross) were lyzed by gentle stirring in 3% octylglucoside, 20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 2 mM CaCl2 at 4xc2x0 C. for 2 h. The lysate was centrifuged at 100,000 g for 1 h. The supernatant obtained was applied to a 5 mL lentil lectin sepharose 4B column (E.Y. Labs) preequilibrated with 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl2, 1% octylglucoside (buffer A). After 2 h incubation, the column was washed with 50 mL cold buffer A. The lectin-retained GPIIb-IIIa was eluted with buffer A containing 10% dextrose. All procedures were performed at 4xc2x0 C. The GPIIb-IIIa obtained was  greater than 95% pure as shown by SDS polyacrylamide gel electrophoresis.
Incorporation of GPIIb-IIIa in Liposomes
A mixture of phosphatidylserine (70%) and phosphatidylcholine (30%) (Avanti Polar Lipids) were dried to the walls of a glass tube under a stream of nitrogen. Purified GPIIb-IIIa was diluted to a final concentration of 0.5 mg/mL and mixed with the phospholipids in a protein:phospholipid ratio of 1:3 (w:w). The mixture was resuspended and sonicated in a bath sonicator for 5 min. The mixture was then dialyzed overnight using 12,000-14,000 molecular weight cutoff dialysis tubing against a 1000-fold excess of 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl2 (with 2 changes). The GPIIb-IIIa-containing liposomes were centrifuged at 12,000 g for 15 min and resuspended in the dialysis buffer at a final protein concentration of approximately 1 mg/mL. The liposomes were stored at xe2x88x9270xc2x0 C. until needed.
Competitive Binding to GPIIb-IIIa
The binding to the fibrinogen receptor (GPIIb-IIIa) was assayed by an indirect competitive binding method using [3H]-SKandF-107260 as an RGD-type ligand. The binding assay was performed in a 96-well filtration plate assembly. (Millipore Corporation, Bedford, Mass.) using 0.22 um hydrophilic durapore membranes. The wells were precoated with 0.2 mL of 10 xcexcg/mL polylysine (Sigma Chemical Co., St. Louis, Mo.) at room temperature for 1 h to block nonspecific binding. Various concentrations of unlabeled benzadiazapines were added to the wells in quadruplicate. [3H]-SKandF-107260 was applied to each well at a final concentration of 4.5 nM, followed by the addition of 1 xcexcg of the purified platelet GPIIb-IIIa-containing liposomes. The mixtures were incubated for 1 h at room temperature. The GPIIb-IIIa-bound [3H]-SKandF-107260 was seperated from the unbound by filtration using a Millipore filtration manifold, followed by washing with ice-cold buffer (2 times, each 0.2 mL). Bound radioactivity remaining on the filters was counted in 1.5 mL Ready Solve (Beckman Instruments, Fullerton, Calif.) in a Beckman Liquid Scintillation Counter (Model LS6800), with 40% efficiency. Nonspecific binding was determined in the presence of 2 xcexcM unlabeled SKandF-107260 and was consistently less than 0.14% of the total radioactivity added to the samples. All data points are the mean of quadruplicate determinations.
Competition binding data were analyzed by a nonlinear least-squares curve fitting procedure. This method provides the IC50 of the antagonists (concentration of the antagonist which inhibits specific binding of [3H]-SKandF-107260 by 50% at equilibrium). The IC50 is related to the equilibrium dissociation constant (Ki) of the antagonist based on the Cheng and Prusoff equation: Ki=IC50/(1+L/Kd), where L is the concentration of [3H]-SKandF-107260 used in the competitive binding assay (4.5 nM), and Kd is the dissociation constant of [3H]-SKandF-107260 which is 4.5 nM as determined by Scatchard analysis.
Preferred compounds of this invention have an affinity for the vitronectin receptor relative to the fibrinogen receptor of greater than 3:1. More preferred compounds have a ratio of activity of greater than 10:1. The comparative results of enhanced binding of the compounds of this invention to the vitronecton receptor relative to the fibrinogen receptor are given in Table 1 below:
Vascular Smooth Muscle Cell Migration Assay
The compounds of the instant invention were tested for their ability to inhibit the migration and proliferation of smooth muscle tissue in an artery or vein in order to assess their ability to prevent restenosis of an artery, such as that which typically occurs following angioplasty.
Rat or human aortic smooth muscle cells were used. The cell migration was monitored in a Transwell cell culture chamber by using a polycarbonate membrane with pores of 8 um (Costar). The lower surface of the filter was coated with vitronectin. Cells were suspended in DMEM supplemented with 0.2% bovine serum albumin at a concentration of 2.5-5.0xc3x97106 cells/mL, and were pretreated with test compound at various concentrations for 20 min at 20xc2x0 C. The solvent alone was used as control. 0.2 mL of the cell suspension was placed in the upper compartment of the chamber. The lower compartment contained 0.6 mL of DMEM supplemented with 0.2% bovine serum albumin. Incubation was carried out at 37xc2x0 C. in an atmosphere of 95% air/5% CO2 for 24 hr. After incubation, the non-migrated cells on the upper surface of the filter were removed by gentle scraping. The filter was then fixed in methanol and stained with 10% Giemsa stain. Migration was measured either by a) counting the number of cells that had migrated to the lower surface of the filter or by b) extracting the stained cells with 10% acetic acid followed by determining the absorbance at 600 nM.